The mechanism by which a completed peptide is separated from transfer RNA (protein chain termination) is not yet understood. We have published data that suggest the process may involve an enzymatic activity different from the ribosomebound peptidyl transferase. Our experimental approach is studying the properties of cell-free extracts of E. coli, both normal and mutants, that are resistant to antibiotics which interfere with protein chain elongation or termination. The enzyme peptidyl-tRNA hydrolase is not required for protein chain termination. Its metabolic role is concerned with scavenging peptidyl-tRNA that is ejected from ribosomes during protein biosynthesis. A mutant E. coli has been isolated which has a temperature-sensitive hydrolase. Studies on the physiology of this mutant reveal an essential role in protein metabolism for peptidyl-tRNA hydrolase. If the enzyme is inactivated by high temperature, peptidyl-tRNA accumulates in the mutant cells and they die, unable to form a colony after return to a low permissive temperature. The nature of released peptidyl-tRNA will be studied in the proposed research. The rate at which various tRNA's are converted to released peptidyl-tRNA will be measured. Further studies of the physiology and genetics of the mutant will be aimed at the mechanism of inhibition of protein biosynthesis at high temperature. The properties of the purified mutant enzyme will also be studied.